| 1. |
- Holmér, Andreas, et al.
(författare)
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The factor H variant associated with age-related macular degeneration (H384) and the non-disease associated form bind differentially to C-reactive protein, fibromodulin, DNA and necrotic cells.
- 2007
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 282:15, s. 10894-10900
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Tidskriftsartikel (refereegranskat)abstract
- Recently, a polymorphism in the complement regulator factor H (FH) gene has been associated with age-related macular degeneration. When histidine instead of tyrosine is present at position 384 in the seventh complement control protein (CCP) domain of FH, the risk for age-related macular degeneration is increased. It was recently shown that these allotypic variants of FH, in the context of a recombinant construct corresponding to CCPs 6 - 8, recognize polyanionic structures differently, which may lead to altered regulation of the alternative pathway of complement. We show now that His-384, corresponding to the risk allele, binds C-reactive protein (CRP) poorly compared with the Tyr-384 form. We also found that C1q and phosphorylcholine do not compete with FH for binding to C-reactive protein. The interaction with extracellular matrix protein fibromodulin, which we now show to be mediated, at least in part, by CCP6 - 8 of FH, occurs via the polypeptide of fibromodulin and not through its glycosaminoglycan modifications. The Tyr-384 variant of FH bound fibromodulin better than the His-384 form. Furthermore, we find that CCP6 - 8 is able to interact with DNA and necrotic cells, but in contrast the His-384 allotype binds these ligands more strongly than the Tyr-384 variant. The variations in binding affinity of the two alleles indicate that complement activation and local inflammation in response to different targets will differ between His/His and Tyr/Tyr homozygotes.
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| 2. |
- Okroj, Marcin, et al.
(författare)
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Antibodies against Kaposi sarcoma-associated herpes virus (KSHV) complement control protein (KCP) in infected individuals
- 2007
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Ingår i: Vaccine. - : Elsevier BV. - 1873-2518 .- 0264-410X. ; 25:48, s. 8102-8109
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Tidskriftsartikel (refereegranskat)abstract
- Kaposi sarcoma-associated herpesvirus (KSHV) is the most important etiopathological factor of Kaposi's sarcoma (KS) and some specific types of malignant lymphomas. One of the viral lytic genes encodes the KSHV complement control protein (KCP), which functionally mimics human complement inhibitors. Although this protein provides an advantage for evading the complement attack, it can serve as target for adaptive immune response. Herein, we identified anti-KCP IgG antibodies in patients with KS and KSHV-related lymphomas. KCP-specific antibodies were only detected in sera of those patients who had high titres of antibodies against lytic or latent KSHV antigens. Complement control protein domain 2 (CCP2) was found to be the most immunogenic part of the KCP protein. Furthermore, pre-incubation of KCP-expressing CHO cells with patient sera containing anti-KCP antibodies resulted in an increased complement deposition when incubated with human serum.
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| 3. |
- Okroj, Marcin, et al.
(författare)
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Prevalence of antibodies against Kaposi's sarcoma associated herpes virus (KSHV) complement inhibitory protein (KCP) in KSHV-related diseases and their correlation with clinical parameters.
- 2011
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Ingår i: Vaccine. - : Elsevier BV. - 1873-2518 .- 0264-410X. ; 29, s. 1129-1134
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Tidskriftsartikel (refereegranskat)abstract
- Kaposi's sarcoma-associated herpes virus (KSHV) encodes its own inhibitor of the complement system, designated KSHV complement control protein (KCP). Previously, we detected anti-KCP antibodies in a small group of 22 patients suffering from Kaposi's sarcoma (KS) and KSHV-related lymphoproliferative diseases (Vaccine, 25:8102-9). Anti-KCP antibodies were more prevalent in individuals suffering from KSHV-related lymphomas than KS and also in those with high titer of antibodies against lytic KSHV antigens. Herein we analyze anti-KCP antibodies in 175 individuals originating from three different groups from northern Sweden or Italy, which included patients suffering from classical or HIV-associated KS, Multicentric Castleman's Disease, KSHV-associated solid lymphoma, pleural effusion lymphoma and healthy individuals with detectable KSHV immune response. Our current study confirmed previous observations concerning antibody prevalence but we also analyzed correlations between anti-KCP antibodies and classical KS evolution, clinical stage and viral load in body fluids. Furthermore, we show that patient's anti-KCP antibodies are able to decrease the ability of KCP to inhibit complement. This fact combined with results of statistical analysis suggests that KCP inactivation by specific antibodies may influence progression of classical KS.
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| 4. |
- Blom, Anna, et al.
(författare)
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Mutations in alpha-chain of C4BP that selectively affect its factor I cofactor function
- 2003
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 278:44, s. 43437-43442
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Tidskriftsartikel (refereegranskat)abstract
- C4b-binding protein (C4BP) inhibits all pathways of complement activation, acting as a cofactor to the serine protease factor I (FI) in the degradation of activated complement factors C4b and C3b. C4BP is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain, the alpha- and beta-chains being composed of eight and three complement control protein (CCP) domains, respectively. In previous studies we have localized cofactor activity and binding of C4b to alpha-chain CCP1-3 of C4BP, whereas the binding of C3b required additionally CCP4. Likewise, introduced point mutations that decreased binding of C4b/C3b caused a decrease in cofactor activity. In the present study, we describe two mutants of C4BP, K126Q/K128Q and F144S/F149S, clustered on alpha-chain CCP3, which selectively lost their ability to act as cofactors in the cleavage of both C4b and C3b. Both mutants show the same binding affinity for C4b/C3b as measured by surface plasmon resonance and have the same inhibitory effect on formation and decay of the classical pathway C3-convertase as the wild type C4BP. It appears that C4b and C3b do not undergo the same conformational changes upon binding to the C4BP mutants as during the interaction with the wild type C4BP, which then results in the observed loss of the cofactor activity.
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| 5. |
- Blom, Anna, et al.
(författare)
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Structural requirements for the complement regulatory activities of C4BP
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:29, s. 27136-27144
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Tidskriftsartikel (refereegranskat)abstract
- C4b-binding protein (C4BP) is a regulator of the classical complement pathway C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain; the alpha- and beta-chains are composed of eight and three complement control protein (CCP) domains, respectively. To elucidate the importance of the polymeric nature of C4BP and the structural requirements for the interaction between C4b and the alpha-chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally, four variants in which double alanine residues were introduced between CCPs were functionally characterized. The smallest truncated C4BP variant still active in regulating fluid phase C4b comprised CCP1-3. The monomeric variants were less efficient than polymeric C4BP in degrading C4b on cell surfaces. All three N-terminal CCP domains contributed to the binding of C4b and were important for full functional activity; CCP2 and CCP3 were the most important. The spatial arrangements of the first CCPs were found to be important, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 and 3, and CCPs 3 and 4 resulted in functional impairment. The results presented here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expression of full functional activity.
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| 6. |
- Friedrich, Ute, et al.
(författare)
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Structural and energetic characteristics of the heparin-binding site in antithrombotic protein C
- 2001
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 276:26, s. 24122-24128
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Tidskriftsartikel (refereegranskat)abstract
- Human activated protein C (APC) is a key component of a natural anticoagulant system that regulates blood coagulation. In vivo, the catalytic activity of APC is regulated by two serpins, alpha1-antitrypsin and the protein C inhibitor (PCI), the inhibition by the latter being stimulated by heparin. We have identified a heparin-binding site in the serine protease domain of APC and characterized the energetic basis of the interaction with heparin. According to the counter-ion condensation theory, the binding of heparin to APC is 66% ionic in nature and comprises four to six net ionic interactions. To localize the heparin-binding site, five recombinant APC variants containing amino acid exchanges in loops 37, 60, and 70 (chymotrypsinogen numbering) were created. As demonstrated by surface plasmon resonance, reduction of the electropositive character of loops 37 and 60 resulted in complete loss of heparin binding. The functional consequence was loss in heparin-induced stimulation of APC inhibition by PCI, whereas the PCI-induced APC inhibition in the absence of heparin was enhanced. Presumably, the former observations were due to the inability of heparin to bridge some APC mutants to PCI, whereas the increased inhibition of certain APC variants by PCI in the absence of heparin was due to reduced repulsion between the enzymes and the serpin. The heparin-binding site of APC was also shown to interact with heparan sulfate, albeit with lower affinity. In conclusion, we have characterized and spatially localized the functionally important heparin/heparan sulfate-binding site of APC.
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| 7. |
- Korpetinou, Angeliki, et al.
(författare)
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Serglycin Is Implicated in the Promotion of Aggressive Phenotype of Breast Cancer Cells
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:10
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Tidskriftsartikel (refereegranskat)abstract
- Serglycin is a proteoglycan expressed by some malignant cells. It promotes metastasis and protects some tumor cells from complement system attack. In the present study, we show for the first time the in situ expression of serglycin by breast cancer cells by immunohistochemistry in patients' material. Moreover, we demonstrate high expression and constitutive secretion of serglycin in the aggressive MDA-MB-231 breast cancer cell line. Serglycin exhibited a strong cytoplasmic staining in these cells, observable at the cell periphery in a thread of filaments near the cell membrane, but also in filopodia-like structures. Serglycin was purified from conditioned medium of MDA-MB-231 cells, and represented the major proteoglycan secreted by these cells, having a molecular size of similar to 250 kDa and carrying chondroitin sulfate side chains, mainly composed of 4-sulfated (similar to 87%), 6-sulfated (similar to 10%) and non-sulfated (similar to 3%) disaccharides. Purified serglycin inhibited early steps of both the classical and the lectin pathways of complement by binding to C1q and mannose-binding lectin. Stable expression of serglycin in less aggressive MCF-7 breast cancer cells induced their proliferation, anchorage-independent growth, migration and invasion. Interestingly, over-expression of serglycin lacking the glycosaminoglycan attachment sites failed to promote these cellular functions, suggesting that glycanation of serglycin is a pre-requisite for its oncogenic properties. Our findings suggest that serglycin promotes a more aggressive cancer cell phenotype and may protect breast cancer cells from complement attack supporting their survival and expansion.
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| 8. |
- Leffler, Jonatan, et al.
(författare)
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Annexin-II, DNA and histones serve as Factor H ligands on the surface of apoptotic cells.
- 2010
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 285:6, s. 3766-3776
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Tidskriftsartikel (refereegranskat)abstract
- Apoptotic cells are opsonized by complement components such as C1q and C3b, which increases their susceptibility to phagocytosis. Soluble complement inhibitors such as factor H (fH)b also recognise apoptotic cells to minimize the pro-inflammatory effects of downstream complement activation. We used four radiolabelled protein constructs that span different regions of the 20 CCP modules that make up fH, and found that fragments comprising CCPs 6-8, CCPs 8-15 and CCPs 19-20 but not CCPs 1-4, bound to apoptotic Jurkat T-cells. There are four possible ligand types on apoptotic cells that could recruit fH: proteins, carbohydrates, lipids and DNA. We found that CCPs 6-8 of fH bind to annexin-II, a trypsin-insensitive protein that becomes exposed on surfaces of apoptotic cells. The second ligand of fH, which interacts with CCPs 6-8 and 19-20, is DNA. Confocal microscopy showed co-localisation of fH with antibodies specific for DNA. FH also binds to histones devoid of DNA and CCPs 1-4, 6-8 and 8-15 mediate this interaction. Treatment of apoptotic cells with neuraminidase, chondroitinase, heparitinase and heparinase did not change fH-binding. Treatment of apoptotic cells with phospholipase A2 dramatically increased both binding of fH and cell-surface DNA. We also excluded the possibility that fH interacts with lysophospholipids using surface plasmon resonance and flow cytometry with lipid-coated beads. Identification of annexin-II as one of the fH ligands on apoptotic cells together with the fact that autoantibodies against annexin-II are found in systemic lupus erythematosus provides further insight into understanding of the pathogenesis of this disease.
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| 9. |
- Nilsson, Sara, et al.
(författare)
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Mutations in complement factor I as found in atypical hemolytic uremic syndrome lead to either altered secretion or altered function of factor I
- 2010
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Ingår i: European Journal of Immunology. - : Wiley. - 1521-4141 .- 0014-2980. ; 40:1, s. 172-185
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Tidskriftsartikel (refereegranskat)abstract
- The complement system is regulated by inhibitors such as factor I (FI), a serine protease that degrades activated complement factors C4b and C3b in the presence of specific cofactors. Mutations and polymorphisms in FI and its cofactors are associated with atypical hemolytic uremic syndrome (aHUS). All 14 complement factor I mutations associated with aHUS analyzed in this study were heterozygous and generated premature stop codons (six) or amino acid substitutions (eight). Almost all of the mutants were expressed by human embryonic kidney 293 cells but only six mutants were secreted into the medium, three of which were at lower levels than WT. The remaining eight mutants were not secreted but sensitive to deglycosylation with endoglycosidase H, indicating that they were retained early in the secretory pathway. Six secreted mutants were purified and five of them were functionally altered in degradation of C4b/C3b in the fluid-phase in the presence of various cofactors and on endothelial cells. Three mutants cleaved surface-bound C3b less efficiently than WT. The D501N mutant was severely impaired both in solution and on surface irrespective of the cofactor used. in conclusion, mutations in complement factor I affect both secretion and function of FI, which leads to impaired regulation of the complement system in aHUS.
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| 10. |
- Okroj, Marcin, et al.
(författare)
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Functional analyses of complement convertases using c3 and c5-depleted sera.
- 2012
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:10
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Tidskriftsartikel (refereegranskat)abstract
- C3 and C5 convertases are central stages of the complement cascade since they converge the different initiation pathways, augment complement activation by an amplification loop and lead to a common terminal pathway resulting in the formation of the membrane attack complex. Several complement inhibitors attenuate convertase formation and/or accelerate dissociation of convertase complexes. Functional assays used to study these processes are often performed using purified complement components, from which enzymatic complexes are reconstituted on the surface of erythrocytes or artificial matrices. This strategy enables identification of individual interactions between convertase components and putative regulators but carries an inherent risk of detecting non-physiological interactions that would not occur in a milieu of whole serum. Here we describe a novel, alternative method based on C3 or C5-depleted sera, which support activation of the complement cascade up to the desired stages of convertases. This approach allows fast and simple assessment of the influence of putative regulators on convertase formation and stability. As an example of practical utility of the assay, we performed studies on thioredoxin-1 in order to clarify the mechanism of its influence on complement convertases.
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